The major objective of this research program is to provide the applicant with new and intensive training in the molecular and cell biology of ion transport. Using hybridoma and recombinant DNA technologies, Dr. Fambrough's laboratory has recently cloned and sequenced cDNAs encoding the major isoforms of the avian Na+, K+- ATPase, and expressed cDNAs for a- and B- subunits of the sodium pump in tissue cultured mammalian cells. Combining these molecular biological strategies and reagents with his skills in ion transport physiology, the applicant will investigate correlations between the structure and function of the avian Na+, K+-ATPase isoforms. In initial gene transfer studies, a high-resolution, ion-sensitive electrode system will be used to characterize the ion transport parameters and sensitivity to ouabain for each of the major alpha- subunit isoforms expressed in mouse L cells. In addition, the numbers and affinity of ouabain-binding sites will be analyzed for each of the isoforms. Regions of the isoform molecules critical to ion transport and ouabain binding will be identified by in vitro mutagenesis strategies. Chimeric genes will be constructed, transfected into tissue cultured cells, and the expressed sodium pumps studied with isoform-specific monoclonal antibodies and physiological assays. Oligonucleotide-directed mutagenesis of genes encoding given isoforms will be used to identify the specific nucleotide sequences which determine these functions. These studies should provide insights into the molecular basis for the functional diversity of the Na+, K+-ATPase, as well as important structural information regarding the ouabain binding site. Using this comprehensive training in later, independent research, the applicant will investigate structure-function and structure- bioregulation relationships of the Na+, K+-ATPase in renal cells.